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A general process for the development of peptide-based immunoassays for monoclonal antibodies.


ABSTRACT:

Purpose

Monoclonal antibodies (mAb) are an important and growing class of cancer therapeutics, but pharmacokinetic analyses have in many cases been constrained by the lack of standard and robust pharmacologic assays. The goal of this project was to develop a general method for the production of immunoassays that can measure the levels of therapeutic monoclonal antibodies in biologic samples at relevant concentrations.

Methods

Alemtuzumab and rituximab are monoclonal approved for the treatment of B-cell malignancies and were used as a model system. Phage-displayed peptide libraries were screened for peptide sequences recognized by alemtuzumab (anti-CD52) or rituximab (anti-CD20). Synthetic biotinylated peptides were used in enzyme-linked immunosorbent assays (ELISA). Peptides directly synthesized on polymer resin beads were used in an immunofluorescent-based assay.

Results

Peptide mimetope sequences were recovered for both mAb and confirmed by competitive staining and kinetic measurements. A peptide-based ELISA method was developed for each. The assay for rituximab had a limit of detection of 4 microg/ml, and the assay for alemtuzumab had a limit of detection of 1 microg/ml. Antibody-specific staining of peptide conjugated beads could be seen in a dose-dependent manner.

Conclusion

Phage-displayed peptide libraries can be a source of highly specific mimetopes for therapeutic mAb. The biotinylated forms of those peptides are compatible with conventional ELISA methods with sensitivities comparable to other assay methods and sufficient for pharmacological studies of those mAb given at high dose. The process outlined here can be applied to any mAb to enable improved pharmacokinetic analysis during the development and clinical use of this class of therapies.

SUBMITTER: Sanchez AB 

PROVIDER: S-EPMC2921063 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Publications

A general process for the development of peptide-based immunoassays for monoclonal antibodies.

Sanchez Ana B AB   Nguyen Tammy T   Dema-Ala Rhanika R   Kummel Andrew C AC   Kipps Thomas J TJ   Messmer Bradley T BT  

Cancer chemotherapy and pharmacology 20100120 5


<h4>Purpose</h4>Monoclonal antibodies (mAb) are an important and growing class of cancer therapeutics, but pharmacokinetic analyses have in many cases been constrained by the lack of standard and robust pharmacologic assays. The goal of this project was to develop a general method for the production of immunoassays that can measure the levels of therapeutic monoclonal antibodies in biologic samples at relevant concentrations.<h4>Methods</h4>Alemtuzumab and rituximab are monoclonal approved for t  ...[more]

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